Gene trapping is a method of random insertional mutagenesis that permits visualization of trapped gene expression. Since gene trapping can be carried out in mouse embryonic stem (ES) cells, it is theoretically possible to create and permanently store a collection of mutant alleles representing every gene in the mouse genome. ES cells carrying a gene trap insertion can be used to generate the corresponding mouse strain. Thus, such a resource would be invaluable for exploring the expression and function of mammalian genes, individually and collectively, in a convenient model system. Since current evidence suggests that many genes have multiple roles, beyond the ones evident by assessing a null allele, it is highly desirable that the insertion mutations be conditional. In this way, the null genotype could be induced at specific times and places within the animal. The standard methods for identification of trapped genes are RNA-based, utilizing expensive and technically challenging RT-PCR techniques. With the immanent completion of the mouse genome it should be possible to use cheaper and simpler DNA-based techniques to specify the location of gene trap insertions. Another important consideration in the design of a library of gene trap insertions is the strain of mouse from which the ES cell line is derived. The mutations should be generated in a genetic background that is suitable for the widest array of applications. Currently, the most widely used ES cell lines are derived from various 129-derived strains, which are not appropriate for many studies. The specific aims of this proposal are designed to 1) generate and test multifunctional, conditional gene trap vectors, 2) develop robotic, DNA-based methods of identifying gene trap insertions and 3) generate and characterize new ES cell lines with genetic backgrounds compatible with a wide array of studies, including those of the auditory system. Successful completion of these aims will set the stage for large-scale gene trap cell line production, which will, in turn, accelerate the production of mouse models of human genetic disease.